Lack of serotonin1B receptor expression leads to age-related motor dysfunction, early onset of brain molecular aging and reduced longevity

Abstract

Normal aging of the brain differs from pathological conditions and is associated with increased risk for psychiatric and neurological disorders. In addition to its role in the etiology and treatment of mood disorders, altered serotonin (5-HT) signaling is considered a contributing factor to aging; however, no causative role has been identified in aging. We hypothesized that a deregulation of the 5-HT system would reveal its contribution to age-related processes and investigated behavioral and molecular changes throughout adult life in mice lacking the regulatory presynaptic 5-HT(1B) receptor (5-HT(1B)R), a candidate gene for 5-HT-mediated age-related functions. We show that the lack of 5-HT(1B)R (Htr1b(KO) mice) induced an early age-related motor decline and resulted in decreased longevity. Analysis of life-long transcriptome changes revealed an early and global shift of the gene expression signature of aging in the brain of Htr1b(KO) mice. Moreover, molecular changes reached an apparent maximum effect at 18-months in Htr1b(KO) mice, corresponding to the onset of early death in that group. A comparative analysis with our previous characterization of aging in the human brain revealed a phylogenetic conservation of age-effect from mice to humans, and confirmed the early onset of molecular aging in Htr1b(KO) mice. Potential mechanisms appear independent of known central mechanisms (Bdnf, inflammation), but may include interactions with previously identified age-related systems (IGF-1, sirtuins). In summary, our findings suggest that the onset of age-related events can be influenced by altered 5-HT function, thus identifying 5-HT as a modulator of brain aging, and suggesting age-related consequences to chronic manipulation of 5-HT.

Figure 1. Early onset of age-related motor decline and reduced longevity in Htr1b^KO mice

(a and b) Htr1b^KO mice displayed early age-related motor impairment in the rotarod test ((a) latency to fall: F₃,₁₁₇ = 11.37, P < e⁻⁶; genotype effect: F = 15.04, P < 0.0005; genotype*age: F = 2.05, P = 0.11) and the coat hanger test ((b) latency to fall: F₃,₁₁₇ = 8.67, P < 0.0001; genotype effect: F₁,₁₂₀ = 16.29, P < 0.0001; genotype*age: F₃,₁₁₇ = 1.98, P = 0.12). Post hoc tests: *P < 0.05, **P < 0.005. (c and d) Total activity decreased with age in WT and Htr1b^KO mice in the open field (OF) (c) and EPM (d) tests (age-effect. OF: F = 4.12, P = 0.008; EPM: F = 20.78, P < e⁻⁵), although variability in the 12- and 18-month age groups suggested a potential age*genotype interaction (genotype*age-effect. OF: F = 3.09, P = 0.03; EPM: F = 3.4, P = 0.02; all other effects, P > 0.05). Different cohort of mice was used for each time point to avoid memory savings between experiments (a–d: n = 13–18 per group and per age). (e) Procedural learning curves in the rotarod test were essentially similar, but reached lower maximal values in Htr1b^KO mice (see (a)). (f) Kaplan–Meyer survival curves revealed a significant decreased in longevity in Htr1b^KO mice (P < 0.0001). (g) Mortality curves: Htr1b^KO mice displayed a 3.75-fold increased hazard ratio (P < 0.0005). Hatched curve represents Htr1b^KO mortality curve superimposed on the WT curve (f and g, WT, n = 21; KO, n = 24).

Figure 2. Peripheral markers in Htr1b^KO and WT mice

(a) Body weight (n = 14–16 per group, P > 0.05). (b) Colon mucosal and smooth muscle layers and lumen were equivalent in WT and KO mice (hematoxylin/eosin staining; representative sections from old WT and KO groups). (c–e) Serum levels of albumin (c), insulin (d), insulin-like growth factor 1 (IGF-1; e) and IgG (f). (c–e): Young (3 months; WT, n = 5; KO, n = 5) and old (18 months; WT, n = 6; KO, n = 6). Statistical significance: main-age effects for albumin (F₁,₂₂ = 10.2, P = 0.005), insulin (F₁,₂₂ = 11.8, P < 0.005) and IgG (F₁,₂₂ = 21.27, P < 0.001). Main genotype effect for albumin (F₁,₂₂ = 4.4, P = 0.08), genotype*age interaction (F_1,22 = 9.2, P = 0.02). Main genotype effect for insulin (F_1,22 = 5.02, P < 0.05) and age–genotype interaction for IgG (F₃,₂₂ = 6.5, P < 0.05). All other effects, P > 0.05. Post hoc tests; ** P < 0.01, * P < 0.05. Error bars represent s.e.m.

Figure 3. Intact dopaminergic terminal density in old Htr1b^KO mice

(a) Representative photographs of DAT immunohistochemistry in 18-month-old WT and Htr1b^KO mice (n = 4 per group, P > 0.05). (b) DAT immunoreactivity density. (c) DAT striatal area (similar results were obtained at 3, 6 and 24 months of age).

Figure 4. Correlation of age-related gene expression ‘signatures’ in the brains of WT and Htr1b^KO mice, and phylogenetic conservation of age-effect between mice and humans

(a–c) WT/Htr1b^KO age-effect comparison. (a) Venn diagrams of age-related transcript changes in CTX and STR (n = 3–4 arrays per age-, genotype- and brain regions; total, n = ~ 60 arrays). (b) Correlation graphs between age-effects (LogR = log₂Old/Young) in CTX between genes identified only in WT (n = 106, left), only in KO (n = 672, middle) and in both groups (n = 319, right). (c) Correlation levels (r) of age-effects between indicated WT and Htr1b^KO age groups. All P < 0.0001 (d–f) Mouse/human age-effect comparison. (d) Age-effect correlation graph between genes identified in the mouse and for which age-related expression levels of orthologous genes were available in the human CTX. Black dots indicate genes with most conserved age-effects that are likely to support a large proportion of the overall correlation (see (f) and Supplementary Table S3). (e) Correlation levels between age-related transcript changes in WT or Htr1b^KO mouse CTX and two areas of the human prefrontal CTX. All P < 0.005, except WT18 versus BA47, P = 0.01. Correlations were based on age-effect in rodent (P < 0.001) and identifiable human orthologs (WT, n = 88 genes; KO, n = 271 genes). (f) Selected genes with conserved age-effects in CTX between mouse and human. Values are in average log₂ (old/young ratio) (red: increased; blue: decreased). See Supplementary Table S3 for additional genes and details. BA9/47, Brodmann areas 9/47.

Figure 5. Over 98% of age-related genes displayed early onset of age-related trajectories in Htr1b^KO mice

(a–d) Averaged transcripts profiles for genes with onset of age-related effects in KO mice occurring initially at 10 months (‘Early’: 198 increased (a) and 81 decreased (b) genes; 25.6% of age-related genes) or 18 months (‘late’: 670 increased (c) and 126 decreased (d) genes; 72.6% of age-related genes), compared to the onset of behavioral differences (vertical gray arrow). Less than 2% of age-affected genes had similar profiles in WT and KO mice (not shown). Values are in percentage of WT levels. The 3-month-old groups used for array analysis were bred at a different time and analyzed separately. (e) Gfap age-related transcript profiles as an example of ‘late’ pattern (age-effect, P < 0.0005 in WT and KO; *WT/KO at 18 month, P < 0.05). (f) Confirmation of altered Gfap transcript levels by real-time qPCR: Array/qPCR correlation (r = 0.87, P = 0.005). Values are mean ± s.e.m. (g and h) Functional analysis of molecular aging. (g) The 25 most affected color-coded gene groups in WT and Htr1b^KO mice are regrouped in five main functions: translation (blue), inflammation (red), metabolism (green), cell growth (yellow), cellular respiration (gray), miscellaneous (white). Details in Supplementary Table S4. (h) Proportional representation of genes with ‘early’ (10 months) or ‘late’ (18 months) patterns of initial WT–KO differences in age-related trajectories within the main age-related functions. *P < 0.001, difference from expected proportions (white column and hatched bar).

Figure 6. Upregulated Sirt5 gene expression in CTX of Htr1b^KO mice

(a) Microarray (top), qPCR (middle) and in situ hybridization (ISH, bottom) analyses revealed significant increased Sirt5 transcript levels in Htr1b^KO mice, with normalized differences at 24 months of age. Smaller differences by ISH were mostly due to Sirt5 expression throughout the brain, which precluded background subtraction and likely underestimated specific signal differences. All values are in percentage of young WT controls. Genotype effect: array, P < 0.01; qPCR, P < 0.05; ISH, P < 0.01. Pair-wise correlations (array–qPCR–ISH), all r > 0.65, P < 0.05. (b) Representative color-coded photomicrographs of Sirt5 ³⁵S-ISH at 3, 6, 18 and 24 months of age. Color barcode indicates increased signal intensity.